CN103751774B - The recombinant cell lines of stably express CSFV E 2 protein and in the application of preparing in subunit vaccine for swine fever and diagnostic reagent - Google Patents

Abstract

The invention discloses a recombinant cell line stably expressing classical swine fever virus E2 protein and its application in the preparation of classical swine fever subunit vaccine and diagnostic reagent. Specifically, the recombinant cell line expressing the E2 protein of classical swine fever virus is BCSFV-E2, and the cell line is preserved in the General Microorganism Center of the China Committee for the Collection of Microorganisms, and its preservation number is: CGMCC? No. 7719. The hog fever subunit vaccine prepared by the recombinant cell line of the present invention has high safety, good immune effect, easy large-scale production, is not easily affected by exogenous virus pollution or antibodies, and is not affected by maternal antibodies to pig immunity. Moreover, the swine fever subunit vaccine of the invention can induce the production of high-level swine fever virus neutralizing antibodies after immunizing pigs. In addition, the invention also discloses a method for constructing a recombinant mammalian cell line, a method for preparing a subunit vaccine for swine fever, and the application of the antigen expressed by the recombinant cell line in preparing vaccines and diagnostic reagents for preventing swine fever.

Description

Recombinant cell line stably expressing classical swine fever virus E2 protein and its application in preparation of classical swine fever subunit vaccine and diagnostic reagent

technical field

The present invention relates to a recombinant mammalian cell line stably expressing classical swine fever virus E2 protein and its application in the preparation of classical swine fever subunit vaccines and diagnostic reagents. Specifically, the recombinant cell line involved in the present invention is BCSFV-E2, which The deposit number is: CGMCCNo.7719. The invention also discloses a method for preparing the recombinant cell line and the application of the recombinant cell line in preparing a vaccine for preventing swine fever and in preparing reagents for diagnosing or detecting swine fever virus infection. The invention belongs to the technical field of animal vaccines and veterinary biological products.

Background technique

Classical swine fever (CSF), also known as hog cholera (Hogcholera, HC), is an acute febrile fatal disease caused by swine fever virus (HogCholeravirus, HCV or Classical swinefevervirus, CSFV). Widespread, high morbidity and mortality, great harm. The International Office of Epizootics (OIE) previously designated it as a Class A infectious disease, and now it is listed as a notifiable disease, and my country classifies it as a Class I animal disease.

Swine fever is widespread all over the world, and it is extremely harmful to the pig industry. The current effective preventive measure against the disease is vaccination. Among them, the attenuated swine fever vaccine (strain C) successfully developed by Chinese scientists has made outstanding contributions to the prevention and control of swine fever worldwide. The vaccine is still used by many countries including my country. Moreover, on the basis of this attenuated vaccine strain, various forms of attenuated vaccines such as baby rabbit vaccine, spleen-free lynching vaccine, primary cell vaccine and passage cell vaccine have been developed. However, the production of tissue vaccines requires a large number of healthy animals, and the production process is labor-intensive, costly, and has side effects of vaccination. These unfavorable factors have affected the practical application of this type of vaccine. The production of attenuated vaccines by cultured cells is also troubled by many factors. For example, BVDV and antibodies in the serum used for cell culture will interfere with the growth of the virus, it is difficult to increase the titer of virus antigens, and the attenuated vaccines need to be stored in the cold chain. The final use effect is affected. Moreover, all attenuated vaccines are susceptible to the influence of maternal antibodies in the actual combination process, which is also one of the factors leading to immune failure.

CSFV is an enveloped virus with a particle size of about 40-60nm. The virus genome is a single-stranded positive-strand RNA, about 12.3kb in length, containing a large open reading frame (ORF), encoding a large polyprotein with 3898 amino acid residues and a molecular weight of about 438kDa. The polyprotein is processed into 12 kinds of mature viral proteins by the proteases of the virus and the host cell during translation and after translation, including structural proteins and non-structural proteins, among which the structural proteins include C, E0, E1 and E2 proteins. E2 protein is an important envelope glycoprotein of CSFV, also known as gp55, which is the main antigenic protein of the virus. E2 protein can induce the production of virus neutralizing antibody, which is the main immunoprotective antigen of CSFV. It is also an important target protein for the study of swine fever genetic engineering vaccines. In view of the various defects and deficiencies of existing swine fever vaccines, and with the development of modern genetic engineering technology and cell bioengineering technology, many researchers are trying to develop a new type of swine fever vaccine that can overcome the defects of existing vaccines by means of modern molecular biology. . These new swine fever vaccines include viral live vector vaccines, synthetic peptide vaccines, DNA vaccines, subunit vaccines of proteins expressed in Escherichia coli, and subunit vaccines of E2 proteins expressed by insect baculoviruses. Among them, the E2 protein subunit vaccine expressed by insect baculovirus developed by European researchers has been used so far. The immunity of this vaccine is not affected by maternal antibodies, and it can be used for antibody detection and differential diagnosis from virus infection. For example, Hulst [Hulst, etal. Cytotechnology, 20 (1-3): 271-277] immunized pigs with 20 μg of E2 biphasic water-in-oil emulsion expressed by insect cells, which can resist the virulent attack of 100LD50CSFVBrescia strain. The vaccine was approved in Europe in the 1990s and played an important role in European countries' swine fever eradication programs. However, the vaccine is expressed through the insect baculovirus expression system. Compared with the prokaryotic expression system, the expression system can undergo post-translational processing and modification to a certain extent after protein expression. However, there are still some differences from the natural antigen protein structure of the virus, and the folding and modification of the protein after expression is not as good as that of the mammalian cell expression system. Moreover, when the system produces and prepares antigens, the cells will lyse and die after being infected by the virus, which increases the difficulty of downstream purification and other production processes.

In this invention, our research group explored different expression cell lines to express the E2 protein of classical swine fever virus, and finally found a suitable expression cell line, which overcomes the cytotoxic effect of expressing the envelope glycoprotein of classical swine fever virus E2, and establishes a large-scale screening and other technologies. Results The cell line expressing CSFV structural protein E2 protein was successfully prepared. The cell line expresses a high amount of E2 protein and is easy to purify. The antigen protein expressed by cultured cells can be continuously harvested and easy to produce. The expressed E2 protein antigen can induce a good immune response to immunized pigs. The vaccine prepared by expressing the antigen from the cell line can provide a novel and highly effective preventive preparation for the prevention of swine fever. It can play an important role in the prevention and control of swine fever in my country and even in the world.

Contents of the invention

One of the purposes of the present invention is to provide a safe and efficient subunit vaccine for classical swine fever.

The second object of the present invention is to provide a recombinant cell line stably expressing the E2 protein of classical swine fever virus.

The third object of the present invention is to provide a method for constructing the recombinant cell line stably expressing the E2 protein of classical swine fever virus.

The fourth object of the present invention is to apply the recombinant cell line expressing CSFV E2 protein to prepare vaccines to prevent CSF, or use it to prepare reagents for diagnosing or detecting CSFV infection.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The CSF subunit vaccine of the present invention is characterized in that it contains the CSFV E2 protein expressed by a recombinant mammalian cell line stably expressing the CSFV E2 protein and an adjuvant.

In the present invention, preferably, the recombinant mammalian cell line stably expressing the classical swine fever virus E2 protein is constructed by the following method:

(1) Constructing a eukaryotic expression plasmid, the eukaryotic expression plasmid includes inserting a gene sequence encoding the E2 protein of classical swine fever virus;

(2) Transfecting the eukaryotic expression plasmid into BHK-21 cells;

(3) The cells transfected with the plasmid were selected and cultured in the medium supplemented with G418;

(4) Dilute and clone the selected and cultured cells, harvest the cloned cell culture supernatant and cells, detect and compare the expression of CSFV E2 protein, and obtain a recombinant mammalian cell line stably expressing CSFV E2 protein.

Among them, more preferably, the amino acid sequence of the classical swine fever virus E2 protein is shown in SEQIDNo.1, and the gene sequence encoding the classical swine fever virus E2 protein is shown in SEQIDNo. 2.

According to the above method, the present invention has obtained a recombinant mammalian cell line capable of stably expressing the E2 protein of classical swine fever virus, named BCSFV-E2, classified as baby hamster kidney cell (Babyhamster kidney cell), and preserved in China Microorganism Culture Preservation Management Co., Ltd. The General Microbiology Center of the Committee is located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

Further, the present invention also provides the application of the recombinant mammalian cell line or its culture in the preparation of a vaccine drug for preventing swine fever virus disease. and

The application of the recombinant mammalian cell line or its culture in the preparation of reagents for diagnosing or detecting classical swine fever virus infection.

Furthermore, the present invention also provides a method for preparing a classical swine fever subunit vaccine, characterized in that it comprises the following steps:

(1) When the recombinant mammalian cell line BCSFV-E2 of the present invention is normally passaged to 90% full, it is replaced with a low-serum medium with a serum content of 1-2% to continue culturing for 4-6 days;

(2) Harvest the cell culture supernatant and store it at 4°C. The cell supernatant was concentrated by ultrafiltration with a molecular weight cut-off of 50kD until the E2 antigen ELISA titer was 1:160 to 1:320, and the final concentration was 0.02% thimerosal. Obtain vaccine antigen solution;

(3) The vaccine antigen solution and the adjuvant are mixed at a weight ratio of 1:1 and fully emulsified to prepare a water-in-oil-in-water vaccine.

In the present invention, preferably, the adjuvant is MONTANIDEISA206VG.

The recombinant mammalian cell line stably expressing the classical swine fever virus E2 protein prepared by the present invention is easy to cultivate, proliferates rapidly, can expand infinitely, has stable properties, and high protein expression. After the expressed protein and adjuvant are prepared into a vaccine, pigs can be induced to immunize pigs The body produces high-titer CSFV neutralizing antibodies, which can resist CSFV infection.

The beneficial effects of the present invention are as follows:

1. The recombinant cell culture expressing the CSFV E2 protein of the present invention to prepare a vaccine can induce the animal body to produce high-titer neutralizing antibodies against CSFV, and can neutralize the CSFV in vivo or in vitro, preventing the virus from infecting the animal body.

2. The selected expression system of the present invention is BHK-21 cell, and the obtained recombinant cell line BCSFV-E2 has biological characteristics similar to parental cells, which is conducive to the large-scale production of antigenic protein; The protein can obtain the natural conformation and modification processing close to the viral protein in the expression cell, and has good antigenicity; the recombinant cell can be cultured in suspension and fermented at a high density, and is easy to produce in large quantities.

3. The recombinant expression cell line of the present invention can be cultured and expressed in a serum-free medium or a low-serum medium, which can reduce the cost of antigen or vaccine production.

4. The present invention optimizes the gene codon of the antigen protein gene, which is beneficial to increase the expression of the antigen.

5. The oil adjuvant vaccine prepared by using the recombinant expression cell line cell culture of the present invention can induce the animal body to produce high-titer virus neutralizing antibody, the antibody lasts for a long time, and can provide long-term and effective immune protection to the immunized animal.

6. Utilize the vaccine immunization animal prepared by recombinant cell line of the present invention to express antigen to not produce classical swine fever virus E0 protein antibody and nonstructural protein antibody, can utilize detecting classical swine fever virus E0 protein antibody or nonstructural protein antibody to vaccine immunization and virus infection Animals are identified.

7. The swine fever vaccine prepared by the present invention does not contain viral nucleic acid, cannot replicate, has no pathogenicity, has extremely high biological safety, and the vaccine immunity is not interfered and affected by maternal antibodies or existing antibodies.

Description of drawings

Figure 1 is a schematic diagram of the construction of recombinant eukaryotic cell expression plasmids;

Figure 2 is ELISA detection of E2 protein expression in different cloned cells after transfection;

Figure 3 is the ELISA detection of E2 protein expression in different passages of the screened cloned cell lines;

Fig. 4 is the recombinant cell line expressing CSFVE2 protein IFA detection;

A, the 5th passage of cloned cells; B, the 25th passage of cloned cells; C, normal BHK-21 cell control;

Fig. 5 is the ELISA antibody detection of immune porcine serum swine fever virus;

Fig. 6 is the results of indirect ELISA for detection of antibodies to swine fever virus in pig serum by expressing E2 protein in recombinant cell lines as antigens.

detailed description

Hereinafter, the present invention will be described in detail with reference to Examples, which are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements are all within the protection scope of the present invention.

Example 1 Construction and detection of a recombinant cell line stably expressing classical swine fever virus E2 protein

1 Materials and methods

1.1 Plasmids, strains and cells

The eukaryotic expression vector plasmid pCAG-neo, DH5α competent cells and BHK-21 cells are preserved by the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The plasmid extraction kit and RNA extraction kit are products of QIAGEN, DNA Gel recovery kit was purchased from Shanghai Huashun Biotechnology Co., Ltd., G418 was purchased from Gibco Company, trypsin was purchased from Hyclone Company, Reverse TranscriptaseM-MLV﹑PrimeSTARTM ^{HSDNAPolymerase} ﹑SalI﹑XhoI﹑BamHI﹑T4DNA Ligase was purchased from TaKaRa Company, The anti-CSFVE2 protein monoclonal antibody was prepared by our research group, the classical swine fever virus antigen ELISA detection kit was the product of MedianDiagnostics, the classical swine fever virus E2 protein antibody ELISA detection kit was the product of IDEXX company, and FITC-labeled goat anti-mouse IgG was purchased from Zhongshan Jinqiao Biotechnology, HDTransfectionReagent transfection kit was purchased from Roche Company.

1.2 Construction of recombinant expression plasmids

The opti-CSFV-E2 gene is a gene encoding CSFVE2 protein optimized by eukaryotic tropism codons. The amino acid sequence of the CSFVE2 protein refers to the protein sequence of the CSFVShimen strain (GenBank: AAC68902.2), and the 258th amino acid V of the E2 protein in the protein sequence is modified to amino acid I, and the C-terminal transmembrane sequence is removed to obtain The amino acid sequence of the E2 protein is shown in SEQ ID NO.1. When designing the gene, a Kozak sequence, a SacI restriction site and a protective base were added before the start codon; a terminator, an XhoI restriction site and a protective base were added at the coding end of the E2 protein gene. The opti-CSFV-E2 gene and the gene-specific amplification primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The size of the synthesized gene opti-CSFV-E2 was 1089bp, and both ends contained SacI﹑XhoI restriction sites. The synthetic gene Cloned in the pUC57 plasmid, the sequence is shown in SEQ ID NO.2. The eukaryotic expression vector pCAG-neo was digested with SacI﹑XhoI, then ligated with the opti-CSFV-E2 gene recovered by SacI and XhoI under the action of T4DNA ligase, transformed into DH5α competent cells, and coated with ampicillin After culturing overnight, pick a single colony to amplify and culture and extract the plasmid, and use SacI﹑XhoI double enzyme digestion to identify it; the positive plasmid identified by enzyme digestion is named pCAGneo-opti-CSFV-E2, and sent to the biological company for further analysis Sequencing verification. The schematic diagram of plasmid construction is shown in Figure 1.

1.3 Cell transfection and screening

Select BHK-21 cells in a good growth state to digest and pass on to a 24-well plate. When the BHK-21 cells grow to 90% full, follow the HDTransfectionReagent Transfection Kit Operating Instructions Use the recombinant plasmid pCAGneo-opti-CSFV-E2 to transfect cells. After 48 hours of transfection, a selective medium containing G418 (1000 μg/mL) was added for pressurized culture. After 4 days, the cells were digested with trypsin, passaged in a 96-well plate by the limiting dilution method and continued to be cultured. After 5 days, the cells were observed under an inverted microscope. Clonal number of cells per well. The wells containing one cell colony (that is, one cell cluster) were selected and expanded in 24-well plates, 6-well plates, and cell culture flasks, and the cells were identified by IFA and the expressed proteins were detected by ELISA. Cell clones with strong IFA signal and high antigen expression were screened.

1.4 ELISA detection of cloned cells expressing E2 protein

After the cloned cells were cultured in a 24-well plate for 48 hours, the supernatant was collected. Using the culture supernatant of untransfected BHK-21 cells as a control, the E2 protein in the culture medium of the cloned cell supernatant was detected with a CSFV antigen detection kit, ELISA The detection was carried out according to the instructions of the kit, the OD450 value was measured, and the relative quantitative comparison and screening of the antigen expressed by each cell clone was carried out.

1.5 Indirect immunofluorescence assay to detect the expression of target protein in transfected cells

Inoculate the cloned cells into 24-well or 12-well plates, remove the culture medium after 24 hours, wash 3 times with PBS, fix with 4% paraformaldehyde for 10 minutes, wash 3 times with PBS, act on the cells with PBS containing 0.1% TritonX100 for 10 minutes, PBS Wash 3 times, block with PBS containing 4% BSA for 2 hours, wash once with PBS, add anti-E2 protein monoclonal antibody diluted 1:500, incubate overnight at 4°C, wash 3 times with PBS, add FITC diluted 1:200 Label goat anti-mouse IgG, incubate at room temperature for 2 hours, wash with PBS three times, and observe the results under a fluorescent microscope.

1.6 RT-PCR identification of cloned cells

Extract total cellular RNA with an RNA extraction kit, take 10 μL for reverse transcription, add OligodT 1 μL, RNase inhibitor 1 μL, M-MLV5×Buffer 5 μL, M-MLV transcriptase 1 μL, dNTP (10 mM) 2.5 μL, add double distilled water to 25 μL, mixed, heated at 42°C for 60 minutes, and terminated at 95°C for 5 minutes. Then use opti-CSFV-E2 gene-specific primers to amplify the target gene by PCR.

1.7 Detection of Expression Stability of Recombinant Cell Lines

To test the genetic stability of recombinant cell lines and the stability of expressed antigens. The cell clones selected by comparison were subcultured, and cells of different passages were taken for ELISA detection of E2 protein expression. At the same time, E2 protein-specific monoclonal antibody immunofluorescence staining was performed on the 5th and 25th generation cells, supplemented by nuclear staining, and the proportion of positive cells was observed under a fluorescence microscope.

2 results

2.1 Construction of recombinant expression plasmids

The structural diagram of the constructed recombinant expression plasmid pCAGneo-opti-CSFV-E2 is shown in FIG. 1 . After the extracted recombinant plasmid was digested by XhoΙ﹑SacΙ, it was analyzed by agarose gel electrophoresis, and two bands were found. The sequence between the sites is completely consistent with the designed gene encoding E2 protein.

2.2Establishment of stable expression cell line of E2 gene

2.2.1 Screening of transfected cell lines

After 48 hours of cell transfection, G418 selection medium was added, and the culture was continued in a 96-well plate by the limiting dilution method. After 7 days, clones containing a single cell colony were selected under an inverted microscope and identified by IFA, and 5 positive cells were screened out. clone.

2.2.2 RT-PCR identification of cloned cells

After using RT-PCR to amplify the target gene of the positive cell clones identified by IFA, the results show that the target bands with the same theoretical size can be amplified for the cells of different generations of the screened cell clones. It indicates that the target gene has been stably fused in the cell genome and is genetically stable.

2.3.3 ELISA detection and screening of expressing cell lines

According to the strength of the signal detected by immunofluorescence and the ELISA detection results of the expression of E2 antigen in the cell culture supernatant, 15 cloned cells were initially selected for further comparison with the ELISA detection of the CSFVE2 protein antigen in the culture supernatant, and the results showed that the cells screened by IFA Among the 15 clones, No. 12 clone had the highest OD value (Fig. 2), and No. 12 cell clone was selected for subculture and further characteristic analysis. The CSFVE2 protein ELISA test was performed on the culture supernatant of No. 12 cloned cells after different passages, and the results showed that the screened cloned cell line still maintained a good protein expression level after multiple passages (Figure 3). The results showed that the constructed recombinant cell line could stably express the target gene.

The obtained No. 12 clone capable of stably expressing the E2 protein of classical swine fever virus was named BCSFV-E2, and it was preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. The strain preservation number is: CGMCCNo. June 18th.

2.3.4 Indirect immunofluorescence assay to detect the expression of E2 protein in transfected cells

The indirect immunofluorescence test showed that the cell clones could present a strong yellow-green fluorescence signal at the first generation after cloning, while the control cells did not present a fluorescence signal. Moreover, when the cell clone was subcultured to the 25th generation, it could still stably express the target protein ( FIG. 4 ). As shown in Figure 4, after IFA detection of the 5th generation cells and 25th generation cells, the positive cell rate in the visible field of view was 100%, indicating that the constructed and screened cell lines were genetically stable.

Example 2 Cell line expressing recombinant protein to the immune protection test of pig

Vaccine preparation: When the recombinant mammalian cell line BCSFV-E2 cells (CGMCC No.7719) constructed and screened in Example 1 grow to 90% full after normal passage, change the low serum medium (serum content is 1-2%) and continue Cultivate for 4-6 days, harvest the cell culture supernatant and store it at 4°C. The cell supernatant is concentrated by ultrafiltration with a molecular weight cut-off of 50kD until the ELISA titer of E2 antigen is 1:160 to 1:320, and the final concentration is 0.02% thimerosal. After that is the vaccine antigen solution. The vaccine antigen solution and MONTANIDEISA206VG adjuvant were mixed and fully emulsified at a weight ratio of 1:1 to prepare a water-in-oil-in-water vaccine. The attenuated vaccine in the control group was a commercially available swine fever cell vaccine (derived from primary bovine testicular cells).

Animal grouping and immunization: before immunization, all pigs used in the experiment were blood-separated and serum was tested negative for CSFV antibody. 15 weaned Landrace piglets of similar age were selected and randomly divided into 3 groups: the first group of 5 pigs was the attenuated vaccine immunization control group, and 5 CSFV attenuated vaccines were immunized per pig, and only immunized once (attenuated vaccine group); The group of 5 pigs is a vaccine group prepared by expressing the E2 antigen of the recombinant mammalian cell line BCSFV-E2 (CGMCCNo.7719), immunized with 2ml of the vaccine, and boosted once four weeks after the first immunization (recombinant subunit vaccine group); the third 5 pigs in the same group were the blank control group, and they were injected with PBS as the control group (PBS control group).

Serum neutralizing antibody detection: The experimental pigs were blood-collected once before immunization, and all pig sera were negative for CSFV antibodies by ELISA. Four weeks after the first immunization, the oil vaccine immunization group received a booster immunization. Blood was collected 2 weeks and 4 weeks after the first immunization to separate serum. Blood was collected at 2, 4, 8, 12, 16, 20, 24, and 28 weeks after the second immunization to separate serum. The pig sera of the experimental animals were all tested for CSFV virus neutralizing antibody, and the method for detecting neutralizing antibody was the NPLA method recommended by the OIE manual. The specific method is as follows: after the porcine serum of each group was inactivated at 56° C. for 30 minutes, it was diluted 10 times with DMEM in a 96-well cell plate, and then diluted 2 times sequentially. 50 μL of the diluted serum was mixed with an equal volume of CSFV virus (Shimen strain, 200 TCID ₅₀ /0.1 ml). Incubate at 37°C for 1 hour, then add 100 μL PK-15 cells, culture at 37°C for 3 days, and fix the cells with 20% acetone in PBS. Anti-CSFVE2 protein monoclonal antibody was used as primary antibody, HRP-labeled goat anti-mouse enzyme-labeled antibody was used as secondary antibody, and ACE was used for color development. Three replicates were performed for each serum sample. At the same time, a blank control was established. After the color development of the cell plate, the judgment result was observed under the light microscope. The neutralization titer of the serum is defined as the maximum serum dilution factor that reduces the infection by more than 50%.

Serum ELISA antibody detection: The pig serum collected before and after immunization was tested with IDEXX swine fever antibody detection kit, and the detection method was carried out according to the kit instructions. The antibody blocking rate was calculated according to the method in the instructions. Serum was serially diluted to detect serum antibody titer (the highest dilution of serum at which the antibody blocking rate is greater than 40%).

Immune protection experiment: 32 weeks after vaccine immunization, the experimental pigs were tested for virus challenge protection. The experimental method is to subcutaneously inject 1mL (10 ⁶ TCID50) of CSF Shimen strain blood poison to all three groups of experimental pigs under the same conditions, and observe the mental state, appetite and body temperature of the experimental pigs every day after inoculation. Continue to observe for 14 days.

Results: The serum neutralizing antibody detection results showed that the vaccine prepared by the antigen expressed by the recombinant BHK cell line BCSFV-E2 expressing CSFV E2 protein constructed and prepared by the present invention can induce pigs to produce CSFV virus neutralizing antibodies after immunizing pigs. Two weeks after the second immunization (6 weeks after the primary immunization), the neutralizing antibody reached the peak of 20480, which was significantly higher than the neutralizing antibody level of the attenuated vaccine immunization group. Four weeks after the second immunization (8 weeks after the primary immunization), the recombinant subunit vaccine could induce a titer of 10240 neutralizing antibodies. Afterwards, neutralizing antibodies showed a slow downward trend, but still maintained a high level of neutralizing antibodies. By 7 months (28 weeks) after immunization, the virus neutralizing antibody level of the vaccine immune group prepared by the present invention still reached 640, far higher than the virus neutralizing antibody level (40-50) required to provide immune protection to pigs . In the experiment, the virus neutralizing antibody level of the unimmunized control pig was less than 10 during the whole experiment. The specific experimental data are shown in Table 1.

The results of blocking ELISA to detect antibodies showed that pigs could be induced to produce classical swine fever specific antibodies (antibody blocking rate greater than 40%) 2 weeks after the pig was immunized with the recombinant subunit vaccine of the present invention, and then the antibody level gradually increased. 6-8 weeks to reach the peak. During the duration of the antibody, the antibody level of the recombinant subunit vaccine immunized group was higher than that of the attenuated vaccine group. This high level of antibody persisted for 32 cycles after immunization (Fig. 5).

The results of the virus challenge immune protection experiment of pigs showed that the pigs of the recombinant subunit vaccine and the attenuated vaccine immunization group of the present invention had normal mental state, good appetite and normal body temperature (no more than 41.0°C) during the whole observation period after inoculation. Pigs were disease-free and all survived. However, pigs in the non-immunized control group began to show symptoms such as listlessness, lethargy, and loss of appetite about 3 days after receiving the poison. Increased body temperature (all above 41.0°C). All pigs in the non-immunized group died on the 9th to 11th day after inoculation. Necropsy was carried out on the dead pigs, and typical pathological changes of classical swine fever appeared in the spleen, kidney, bladder, tonsil and other organs. However, pigs in the recombinant subunit vaccine group and the attenuated vaccine immunized group had no pathological changes in relative organs after necropsy.

Table 1 Detection results of virus neutralizing antibody in serum of pigs immunized with recombinant subunit vaccine

^a Recombinant antigen vaccine group received a booster immunization 4 weeks after the initial immunization.

Embodiment 3 uses recombinant cell line to express antigen indirect ELISA method to detect swine fever virus antibody

The BCSFV-E2 cells were expanded and cultured, and the cell culture supernatant was harvested. The cell supernatant was centrifuged at low speed to remove impurities such as cell debris, and then filtered through a 0.45 μm filter membrane for clarification. The clarified supernatant was then concentrated by ultrafiltration with a molecular weight cut-off of 50kD. After being concentrated by about 40 times of volume, it is centrifuged at 12000rpm to remove insoluble impurities. After concentration, the supernatant was dialyzed with pH 8.0 Tris-HCl buffer, combined with DEAE anion resin chromatography column, washed and eluted with pH 8.0 Tris-HCl, 250mM NaCl buffer itd, and the active peak was collected protein. After being concentrated by ultrafiltration, it was chromatographed by molecular sieve chromatography to collect the active peak protein, measure the protein concentration, and store it at -70°C for future use. Purified CSFV-E2 protein was used as the coating antigen to coat a 96-well polystyrene microtiter plate. Dilute the antigen with pH 9.6, 0.1M carbonate buffer to a final concentration of 2 μg/ml, add 100 μl/well to the microtiter plate, and coat overnight at 4°C. Then wash the plate with PBST (PBS + 0.05% Tween) for 3 times; seal the plate with PBST containing 1% BSA, block at 37°C for 2 hours, wash the plate with PBST for 3 times after sealing, and use it immediately for detection or - Store at 20°C for later use.

Antibody detection operation procedure: add the pig serum to be tested (100-fold dilution of the qualitative detection serum, double dilution of the antibody titer detection serum, and set up a positive serum control, a negative serum control, and a blank control without serum), and incubate at 37°C for 1h , washed 3 times with PBST washing solution, 3 minutes each time; added horseradish peroxidase-labeled goat anti-pig IgG (Goat-anti-pigIgG-HRP), incubated at 37°C for 1 hour, washed 4 times with PBST washing solution, 3 minutes each time Minutes; add HRP chromogenic substrate (TMB chromogenic reagent), incubate at room temperature for 5-15 minutes to observe the color reaction; after sufficient color development, add 2M sulfuric acid to stop the color reaction; use a microplate reader to measure the absorbance at 450nm wavelength; judgement result.

When judging the results: the absorbance value of the blank control and negative serum wells is less than or equal to 0.3, and the result is valid when the absorbance value of the positive serum control wells is greater than 0.4; calculate the P/N value = (OD value of the detection well - OD value of the blank control well) / (negative Serum OD value - OD value of blank control well), when the P/N value is equal to or greater than 2, it is positive; the antibody titer of the sample serum is determined by the maximum dilution of the positive serum.

Select 20 pig sera that are known to be positive for CSFV antibodies and 20 pig sera that are negative, and use the purified CSFV-E2 protein as the coated antigen for indirect ELISA detection to test the antigen detection effect. The test results are shown in Figure 6. The OD450 values of all positive sera were greater than 0.5, and the OD450 values of all negative sera were less than 0.3. It shows that this antigen has good specificity and sensitivity.

Claims (6)

1. A subunit vaccine of classical swine fever, characterized in that it contains the classical swine fever virus E2 protein and adjuvant expressed by a recombinant mammalian cell line expressing the classical swine fever virus E2 protein; Described swine fever subunit vaccine is prepared through the following steps: (1) When the recombinant mammalian cell line BCSFV-E2 grows to 90% full after normal passage, change the low serum medium with a serum content of 1-2% and continue to culture for 4-6 days; (2) Harvest the cell culture supernatant and store it at 4°C. The cell supernatant was concentrated by ultrafiltration with a molecular weight cut-off of 50kD until the E2 antigen ELISA titer was 1:160 to 1:320, and the final concentration was 0.02% thimerosal. Obtain vaccine antigen solution; (3) The vaccine antigen solution and the adjuvant are mixed and fully emulsified at a weight ratio of 1:1 to prepare a water-in-oil-in-water vaccine; Wherein, the recombinant mammalian cell line BCSFV-E2 is preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms, and its strain preservation number is: CGMCC No.7719. 2. A recombinant mammalian cell line stably expressing the E2 protein of classical swine fever virus, characterized in that the cell line is named BCSFV-E2, and is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and its strain preservation number is: CGMCCNo .7719. 3. the application of recombinant mammalian cell line or its culture described in claim 2 in the preparation of vaccine medicine for preventing swine fever virus disease. 4. the recombinant mammalian cell line described in claim 2 or the application of its culture in preparation diagnosis or detection swine fever virus infection reagent. 5. A method for preparing a classical swine fever subunit vaccine, characterized in that it may further comprise the steps: (1) When the recombinant mammalian cell line BCSFV-E2 described in claim 2 grows to 90% full after normal passage, the low serum medium that changes serum content is 1～2% continues to cultivate 4-6d; (2) Harvest the cell culture supernatant and store it at 4°C. The cell supernatant was concentrated by ultrafiltration with a molecular weight cut-off of 50kD until the E2 antigen ELISA titer was 1:160 to 1:320, and the final concentration was 0.02% thimerosal. Obtain vaccine antigen solution; (3) The vaccine antigen solution and the adjuvant are mixed at a weight ratio of 1:1 and fully emulsified to prepare a water-in-oil-in-water vaccine. 6. The method according to claim 5, characterized in that the adjuvant is MONTANIDEISA206VG.